Identification of an endocytic signal essential for the antiviral action of IFITM3

Abstract

Members of the interferon-induced transmembrane (IFITM) protein family inhibit the entry of a wide range of viruses. Viruses often exploit the endocytosis pathways to invade host cells and escape from the endocytic vesicles often in response to low pH. Localization to these endocytic vesicles is essential for IFITM3 to interfere with the cytosolic entry of pH-dependent viruses. However, the nature of the sorting signal that targets IFITM3 to these vesicles is poorly defined. In this study, we report that IFITM3 possesses a YxxΦ sorting motif, i.e. 20-YEML-23, that enables IFITM3 to undergo endocytosis through binding to the μ2 subunit of the AP-2 complex. IFITM3 accumulates at the plasma membrane as a result of either mutating 20-YEML-23, depleting the μ2 subunit or overexpressing μ2 mutants. Importantly, blocking endocytosis of IFITM3 abrogates its ability to inhibit pH-dependent viruses. We have therefore identified a critical sorting signal, namely 20-YEML-23, that controls both the endocytic trafficking and the antiviral action of IFITM3. This finding also reveals that as an endocytic protein, IFITM3 first arrives at the plasma membrane before it is endocytosed and further traffics to the late endosomes where it acts to impede virus entry.

Figure 1

The YEML motif regulates the subcellular distribution and endocytosis of IFITM3. A. IFITM3 and its YEML mutations. The 20‐YEML‐23 motif is highlighted in boldface letters. The unfilled boxes denote the two putative membrane domains. B. Effect of mutating the YEML motif on cellular localization of IFITM3. HEK293 cells were transfected with IFITM3 or its YEML mutants, then fed with Alexa 555‐conjugated transferrin at 37°C for 15 min prior to fixation. IFITM3 proteins were stained with anti‐Flag primary antibody and FITC‐conjugated secondary antibody. DAPI was used to stain nuclei. C. IFITM3 undergoes endocytosis. HEK293 cells expressing C‐Flag IFITM3 or the YLAA mutant were incubated with anti‐Flag antibody at 4°C for 30 min in the presence of Alexa 555‐conjugated transferrin. Cells were either fixed directly and stained with anti‐Flag antibody (cell surface expression) or switched to 37°C for 15 min to allow internalization of IFITM3/anti‐Flag antibody complex (antibody uptake). The internalized complexes were then visualized by staining with FITC‐conjugated secondary antibody. To visualize the total IFITM3 or YLAA mutant (total expression), cells were permeabilized with 0.2% saponin prior to immunostaining with anti‐Flag antibody. For wild‐type IFITM3, two exposures of the same image are presented to show the relatively weak staining at the plasma membrane. Representative images are shown. D. Cell surface expression of the N‐ and C‐termini of IFITM3. HEK293 cells were transfected with the N‐ or C‐Myc IFITM3 and the YLAA mutant DNA. Half of the cells were incubated with anti‐Myc antibody at 4°C to stain the N‐ or C‐terminus of IFITM3 at the cell surface. The other half were fixed and permeabilized with 0.2% saponin, followed by anti‐Myc antibody staining for the total expression of IFITM3. Myc‐positive cells were scored by flow cytometry. The ratios of Myc‐positive cells between the cell surface staining and the total expression staining were calculated to determine the relative cell‐surface exposure of the N‐ and C‐termini of IFITM3. The averages of three independent experiments are summarized in the bar graph.

Figure 2

Dynasore and CPZ block IFITM3 endocytosis. HEK293 cells were transfected with Flag‐IFITM3‐Flag plasmid DNA. Twenty‐four hours after transfection, cells were treated with dynasore (160 μM) (A) or CPZ (20 μg ml⁻¹) (B) for different periods of time, followed by immunostaining Flag‐IFITM3. After treatment with dynasore or CPZ, cells were also incubated with Alexa 555‐conjugated transferrin (5 μg ml⁻¹) at 37°C for 10 min to monitor the effectiveness of these treatments on endocytosis. Subcellular localization of Flag‐IFITM3 was determined by staining with anti‐Flag antibody. Nuclei were stained with DAPI. Representative images are shown.

Figure 3

IFITM3 interacts with the μ2 subunit of AP‐2 complex. A. Co‐immunoprecipitation of IFITM3 with the μ subunits. HEK293T cells were transfected with plasmid DNA expressing Flag‐IFITM3 and Myc‐tagged μ subunits. Myc‐μ proteins were immunoprecipitated with anti‐Myc antibody, followed by Western blotting with the indicated antibodies. B. Co‐immunoprecipitation to examine the association of μ2 with IFITM3 mutants that had the YEML sequence altered. C. Co‐immunoprecipitation to detect the interaction of IFITM3 with μ2 mutants WR/AA and FD/AS that bear mutated YxxΦ‐binding pocket. Sizes of protein markers (in kDa) are shown on the left side of each Western blot.

Figure 4

Effect of modulating μ2 level on subcellular distribution of IFITM3. A. The μ2 mutants WR/AA and FD/AS were ectopically expressed in HEK293 cells together with Flag‐IFITM3. Cellular localizations of Flag‐IFITM3 (pseudocoloured as green) and μ2 mutants (pseudocoloured as red) were determined by staining with anti‐Flag and anti‐Myc antibodies respectively. DAPI was used to stain nuclei. Representative images are shown. B. HEK293 cells were transfected with siRNA oligonucleotides targeting μ2. Levels of endogenous μ2 were measured by Western blotting with anti‐μ2 antibody. Tubulin was probed as an internal control. Sizes of protein markers (in kDa) are shown. C. Depletion of μ2 causes accumulation of Flag‐IFITM3 at the plasma membrane. HEK293 cells were transfected with siRNA targeting μ2 and IFITM3 plasmid DNA. Localization of IFITM3 was determined by staining with anti‐Flag antibody. Transferrin internalization was used to monitor the effect of μ2 knock‐down on endocytosis. D. Effect of μ2 knock‐down on subcellular distribution of endogenous IFITM3 in HeLa cells. The endogenous IFITM3 was detected with anti‐IFITM3 antibody. The endocytosis efficiency was monitored by measuring the internalization of transferrin. Representative images are shown.

Figure 5

Mutating the YEML motif diminishes the capacity of IFITM3 to inhibit viruses. A. IAV was used to infect HEK293 cells that stably express IFITM3 or its mutants with altered YEML sequence. Eight hours after infection, cells were lysed and the levels of IAV proteins NP, M2 and HA were determined by Western blotting. Anti‐Flag antibody was used to probe the ectopically expressed IFITM3 and its mutants. Sizes of protein markers are shown on the left side of the Western blots. B. Different doses of IAV were used to infect the IFITM3‐expressing HEK293 cell lines. Eight hours after infection, the infected cells were fixed and stained with anti‐NP antibody followed by flow cytometry analysis to score the infected cells. Results shown are the average of three independent infections. The original flow cytometry data are presented in supplemental http://onlinelibrary.wiley.com/doi/10.1111/cmi.12262/suppinfo. C. Effects of wild‐type IFITM3 and its mutants on infections that were mediated by VSV G protein, Ebola virus GP and the 10A1 protein of MLV. The IFITM3‐expressing HEK293 cells were infected with the MLV‐GFP reporter viruses that were pseudotyped with VSV‐G, EBOV GP or 10A1 proteins. GFP‐positive cells were scored by flow cytometry 40 h after infection. Results shown are the average of three independent experiments. The P‐values were calculated between the data of wild‐type IFITM3 and those of IFITM3 mutants, the numbers are below 0.05 for infections mediated by VSV G and EBOV GP.

Figure 6

Effects of different Y‐motifs on the antiviral activity of IFITM3. A. Conservation of the YEML‐like motif in IFITM3 proteins from different species. The consensus sequence of this Y‐motif is shown. B. hIFITM3‐YERI contains the YERI motif from mouse IFITM3 (mIFITM3), mIFITM3‐YEML has the YEML motif from human IFITM3 (hIFITM3). HEK293 cell lines were generated to stably express these IFITM3 proteins. pQCXIP is the empty retroviral vector and was used to produce the control cell line. Expression levels of IFITM3 proteins were determined by Western blotting. Sizes of protein markers are shown on the left side of the Western blots. C. The IFITM3‐expressing HEK293 cell lines were infected with IAV. Cells were collected 8 h after infection, then immunostained for viral NP protein. The positive cells were scored by flow cytometry. D. Results of three independent infections are summarized in the bar graph.

Figure 7

A model to illustrate the intracellular trafficking of IFITM3 and its antiviral action. Following its synthesis at the ER, IFITM3 traffics to the plasma membrane where its 20‐YEML‐23 motif interacts with the μ2 subunit of AP‐2 complex and undergoes clathrin‐dependent endocytosis. Ubiquitination of K24 is shown. C71/72/105 are palmitoylated. This trafficking mechanism positions IFITM3 on the endocytic pathway that many viruses utilize for cell entry. Examples of these viruses are VSV, IAV and EBOV. MLV is a pH‐independent virus. VSV, vesicular stomatitis virus; IAV, influenza A virus; EBOV, Ebola virus; MLV, murine leukaemia virus. This model is partially adapted from those published in Bailey et al. (2013), Diamond and Farzan (2013) and Perreira et al. (2013).